Project description:Amyloidosis of human islet amyloid polypeptide (hIAPP) is a pathological hallmark of type II diabetes (T2D), an epidemic afflicting nearly 10% of the world's population. To visualize disease-relevant hIAPP fibrils, we extracted amyloid fibrils from islet cells of a T2D donor and amplified their quantity by seeding synthetic hIAPP. Cryo-EM studies revealed four fibril polymorphic atomic structures. Their resemblance to four unseeded hIAPP fibrils varies from nearly identical (TW3) to non-existent (TW2). The diverse repertoire of hIAPP polymorphs appears to arise from three distinct protofilament cores entwined in different combinations. The structural distinctiveness of TW1, TW2 and TW4 suggests they may be faithful replications of the pathogenic seeds. If so, the structures determined here provide the most direct view yet of hIAPP amyloid fibrils formed during T2D.
Project description:Transthyretin-derived amyloidosis (ATTR) is a degenerative, systemic disease characterized by transthyretin fibril deposition in organs like the heart, kidneys, liver, and skin. We report the first cryo-EM structure of transthyretin fibrils isolated from skin tissue of a living patient with a rare genetic mutation (ATTRv F64S). The structure adopts a highly conserved fold previously observed in other ATTR fibrils from different tissues or genetic variants. Mass spectrometry was used to identify common post-translational modifications. The structural consistency between ATTR filaments validates non-invasive skin biopsy as a diagnostic tool.
Project description:Mimivirus 1.2Mb genome is organized into a 30 nm nucleocapsid-like structure made of two closely related GMC-oxidoreductases, also composing the fibrils decorating its virions. In this work, we used MS-proteomics to characterize the protein content of virions and fibrils from different members of the Mimiviridae family (clade A: Mimivirus reunion -Mr- and Mimivirus M4 -M4, clade B: Moumouvirus australiensis -Ma- and Moumouvirus maliensis -Mm, clade C: Megavirus chilensis -Mc- and Megavirus vitis -Mv). Furthermore, we analyzed fractions purified from Mr mutants devoid of one of the two GMC-oxidoreductases (Mr_KOqu_143 and Mr_KOqu_946), or of both GMC-oxidoreductases (Mr_2KO) with or without expression of the GFP fused to the N-terminus of one GMC-oxidoreductase (Mr_2KO-GFP). Our results show the versatility of the protein content of the fibrils, with fibrils composed of different proteins inter- and even intra-clade, clades B and C viruses presenting fibrils with a protein composition closer to each other than that of clade A viruses.
Project description:The calcifying matrix proteins embedded within the shell biomineral structure is a mixture of proteins, glycoproteins, and polysaccharides that are involved in the shell formation processes. Here with develop bottom-up shotgun proteomic investigation of these proteins, thanks to specific transcriptomic data generated from the calcifying mantle of adult Lymnaea.
Project description:Background: An important feature to vitamin D physiology is its gender dependence. The aim of this study was to examine whether vitamin D exerts a sexually dimorphic effect on the blood coagulation pathway among adults with overweight. Methods: This study compared the serum proteomic profiles of age and BMI-matched males (n=26) and pre-menopausal females (n=24) with overweight that attained vitamin D sufficiency after a 12-month intervention. Unprocessed serum was subjected to depletion-free, quantitative proteomic analysis using our previously published methodology. Results. A total of 1,841 proteins were profiled (p < 0.05). The analysed proteins vitamin-K dependent protein C, von Willebrand factor, fibrinogen gamma chain and multimerin-1 were ELISA validated to be differentially affected between genders by vitamin D status improvement. Conclusions: Vitamin D optimization exhibits a sexually dimorphic effect on the blood coagulation pathway among adults with overweight. This gender specific vitamin D effect should be taken into consideration in the design and interpretation of vitamin D observational and intervention studies.
Project description:The microtubule associated protein Tau (MAPT) expressed in neurons is involved in microtubules stabilization, cell morphogenesis and axonal transport. In pathological conditions, Tau assembles into high molecular weight assemblies leading to neuropathological Tau deposits, the hallmark of several neurodegenerative diseases collectively named “tauopathies”, including Alzheimer’s disease. These pathologic Tau assemblies are released by affected neuronal cells and taken up by naïve neighbor cells, propagate from one cell to another and amplify by seeding the aggregation of endogenous Tau. In order to identify plasma membrane proteins exposed extracellularly that interact with extracellularly applied fibrillar Tau assemblies, we exposed pure-neuronal cultures to fibrillar Tau for 10 min, pulled down the associated proteins, and identified them using a proteomic-based approach. Of the six Tau isoforms produced by alternative splicing of the MAPT gene and differing from each other by the presence or absence of one or two inserts in the N-terminal part (0N, 1N or 2N) of the protein and by the presence of either three or four repeated microtubule binding motifs in the protein C-terminal part (3R or 4R), we have expressed and purified 1N3R and 1N4R Tau isoforms and further assembled them into 1N3R and 1N4R Tau fibrils. Using pull-down of whole cell lysates and mass spectrometry, we have identified proteins interacting with extracellularly applied Tau fibrils. We have performed two different experiments with either 1N3R Tau fibrils or 1N4R Tau fibrils (condition 1 and condition 2 respectively). Each condition consists in six experimental replicates of cells exposed 10 min to Tau fibrils and of the non-treated cells used as controls.
Project description:The surface associated proteomics of different P aeruginosa fap genetic variants were evaluated. The bacteria were grown on the glass wool, the biomass was extracted from glass wool and the surface influenced planktonics(SIP)biomass was collected.
Project description:To characterise the abundance of ubiquitylated Tau peptides in the presence of active or inactive variants (wt 196-565 vs. C221A) of the broad spectrum deubiquitylase USP21. Tau peptides are obtained from Tau fibrils purified from post mortem human brains diagnosed with Alzheimer’s. Their detection is facilitated by previous training of the mass spectrometer with in vitro ubiquitylated Tau. The same samples were used to evaluate the percentage of peptides modified with ubiquitin
Project description:SPINT1 is a membrane-anchored serine protease inhibitor that modulates pericellular proteolysis. The pancreas-specific disruption of Spint1 in mice significantly decreased islet size and mass, leading to glucose intolerance, which was causally related to the down-regulation of MafA and insulin. Mechanistically, the silencing of Hepsin (encoding a SPINT1 target protease) counteracted the repressive effect of Spint1 knockdown on MafA and Ins1 expression in β cells. Together, the results suggest that SPINT1 plays a novel role in β cells to maintain glucose homeostasis and insulin production.